Since polypeptides tend to be easily denatured due to their low stability, degraded by proteolytic enzymes in the blood and easily passed through the kidney or liver, protein medicaments, including polypeptides as pharmaceutically effective components, need to be frequently administered to patients to maintain desired blood level concentrations and titers. However, this frequent administration of protein medicaments, especially through injection, causes pain for patients. To solve these problems, many efforts have been made to improve the serum stability of protein drugs and maintain the drugs in the blood at high levels for a prolonged period of time, and thus maximize the pharmaceutical efficacy of the drugs. Pharmaceutical compositions with sustained activity, therefore need to increase the stability of the protein drugs and maintain the titers at sufficiently high levels without causing immune responses in patients.
To stabilize proteins and prevent enzymatic degradation and clearance by the kidneys, a polymer having high solubility, such as polyethylene glycol (hereinafter, referred to simply as “PEG”), was conventionally used to chemically modify the surface of a protein drug. By binding to specific or various regions of a target protein, PEG stabilizes the protein and prevents hydrolysis, without causing serious side effects (Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991). However, despite its capability to enhance protein stability, this PEG coupling has problems such as greatly reducing the number titers of physiologically active proteins. Further, the yield decreases with the increasing molecular weight of the PEG due to the reduced reactivity of the proteins.
Recently, polymer-protein drug conjugates have been suggested. For example, as described in U.S. Pat. No. 5,738,846, a conjugate can be prepared by linking an identical protein drug to both ends of PEG to improve the activity of the protein drug. Also, as described in International Pat. Publication No. WO 92/16221, two different protein drugs can be linked to both ends of PEG to provide a conjugate having two different activities. The above methods, however, were not very successful in sustaining the activity of protein drugs.
On the other hand, Kinstler et al. reported that a fusion protein prepared by coupling granulocyte-colony stimulating factor (G-CSF) to human albumin showed improved stability (Kinstler et al., Pharmaceutical Research 12(12): 1883-1888, 1995). In this publication, however, since the modified drug, having a G-CSF-PEG-albumin structure, only showed an approximately four-fold increase in residence time in the body and a slight increase in serum half-life compared to the single administration of the native G-CSF, it has not been industrialized as an effective long-acting formulation for protein drugs.
An alternative method for improving the in vivo stability of physiologically active proteins is by linking a gene of physiologically active protein to a gene encoding a protein having high serum stability by genetic recombination technology and culturing the cells transfected with the recombinant gene to produce a fusion protein. For example, a fusion protein can be prepared by conjugating albumin, a protein known to be the most effective in enhancing protein stability, or its fragment to a physiologically active protein of interest by genetic recombination (International Pat. Publication Nos. WO 93/15199 and WO 93/15200, European Pat. Publication No. 413,622). A fusion protein of interferon-alpha and albumin, developed by the Human Genome Science Company and marketed under the trade name of ‘Albuferon™’, increased the half-life from 5 hours to 93 hours in monkeys, but it was known to be problematic because it decreased the in vivo activity to less than 5% of unmodified interferon-alpha (Osborn et al., J. Phar. Exp. Ther. 303(2): 540-548, 2002).
On the other hand, an immunoglobulin (Ig) is composed largely of two regions: Fab having an antigen-binding site and Fc having a complement-binding site. Other attempts were made to fuse a protein drug to an immunoglobulin Fc fragment by genetic recombination. For example, interferon (Korean Pat. Laid-open Publication No. 2003-9464), and interleukin-4 receptor, interleukin-7 receptor or erythropoietin (EPO) receptor (Korean Pat. Registration No. 249572) were previously expressed in mammals in a form fused to an immunoglobulin Fc fragment. International Pat. Publication No. WO 01/03737 describes a fusion protein comprising a cytokine or growth factor linked to an immunoglobulin Fc fragment through an oligopeptide linker.
In addition, U.S. Pat. No. 5,116,964 discloses an LHR (lymphocyte cell surface glycoprotein) or CD4 protein fused to an amino terminus or carboxyl terminus of an immunoglobulin Fc fragment by genetic recombination, and U.S. Pat. No. 5,349,053 describes a fusion protein of IL-2 and an immunoglobulin Fc fragment. Other examples of Fc fusion proteins prepared by genetic recombination include a fusion protein of interferon-beta or a derivative thereof and an immunoglobulin Fc fragment (International Pat. Publication No. WO 00/23472), a fusion protein of IL-5 receptor and an immunoglobulin Fc fragment (U.S. Pat. No. 5,712,121), a fusion protein of interferon-alpha and an Fc fragment of an immunoglobulin G4 (U.S. Pat. No. 5,723,125), and a fusion protein of CD4 protein and an Fc fragment of an immunoglobulin G2 (U.S. Pat. No. 6,451,313). Also, as described in U.S. Pat. No. 5,605,690, an Fc variant having an amino acid alteration especially at a complement-binding site or receptor-binding site can be fused to TNF receptor by recombinant DNA technologies to give a TNFR-IgG1 Fc fusion protein. In this way, methods of preparing an Fc fusion protein using an immunoglobulin Fc fragment modified by genetic recombination are disclosed in U.S. Pat. Nos. 6,277,375, 6,410,008 and 6,444,792.
U.S. Pat. No. 6,660,843 discloses a method of producing a conjugate comprising a target protein fused to an immunoglobulin Fc fragment by means of a linker in E. coli by genetic recombination. This method allows the conjugate to be produced at lower cost than when using mammalian expression systems and provides the conjugate in an aglycosylated form. However, since the target protein and the immunoglobulin Fc fragment are produced together in E. coli, if the target protein is glycosylated in nature, it is difficult to apply such a target protein using this method. This method has another problem of expressing the conjugate as inclusion bodies, resulting in very high misfolding rates.
However, such Fc fusion proteins produced by genetic recombination have the following disadvantages: protein fusion occurs only in a specific region of an immunoglobulin Fc fragment, which is at an amino- or carboxyl-terminal end; only homodimeric forms and not monomeric forms are produced; and a fusion could take place only between the glycosylated proteins or between the aglycosylated proteins, and it is impossible to make a fusion protein composed of a glycosylated protein and an aglycosylated protein. Further, a new amino acid sequence created by the fusion may trigger immune responses, and a linker region may become susceptible to proteolytic degradation.
On the other hand, with respect to the development of fusion proteins using an immunoglobulin Fc fragment, there is no report of a conjugate comprising a target protein linked to a human-derived native Fc using a crosslinking agent. The preparation of a conjugate using a linker has the advantages of facilitating the selection and control linking sites and orientation of two proteins to be linked together, and allowing the expression in a monomer, dimer or multimer and the preparation of homologous or heterogeneous constructs. The immunoglobulin Fc fragment can be produced by recombinant DNA technologies using mammalian cells or E. coli. However, to date, there is no report of a native immunoglobulin Fc fragment that is singly mass-produced with high yields in E. coli and applied to long-acting formulations. Also, to date, there has been no attempt for the production of a conjugate comprising a target protein linked to such an E. coli-derived immunoglobulin Fc fragment produced by recombinant DNA technologies by means of a crosslinking agent.
On the other hand, immunoglobulins have antibody functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and sugar moieties present at an Fc fragment of immunoglobulins play important roles in the ADCC and CDC effects (Burton D., Molec. Immun. 22, 161-206, 1985). Immunoglobulins lacking sugar moieties have serum half-lives similar to glycosylated immunoglobulins but 10 to 1000-fold reduced complement and receptor binding affinities (Waldmann H., Eur. J. Immunol. 23, 403-411, 1993; Morrison S., J. Immunol. 143, 2595-2601, 1989).
As described above, a variety of methods have been tried for linking a polymer to a physiologically active protein. Conventional methods enhance the stability of polypeptides but remarkably reduce the activity thereof, or improve the activity of the polypeptides regardless of the stability. Thus, there is a need of a method capable of achieving both minimal activity reduction and stability enhancement for a protein drug.
In this regard, leading to the present invention, the intensive and through research into the development of a long-acting protein drug formulation capable of achieving both minimal activity reduction and stability enhancement, which are conventionally considered difficult to accomplish, resulted in the finding that a protein conjugate, prepared by covalent bond an immunoglobulin Fc fragment, a non-peptide polymer and a physiologically active polypeptide, remarkably extends the serum half-life of the physiologically active protein and maintains higher titers than known protein drugs.